Microbiomes associated with Coffea arabica and Coffea canephora in four different floristic domains of Brazil

Brazilian coffee production relies on the cultivation of Coffea arabica and Coffea canephora. Climate change has been responsible for the decreasing yield of the crops in the country yet the associated microbial community can mitigate these effects by improving plant growth and defense. Although some studies have tried to describe the microorganisms associated with these Coffea species, a study that compares the microbiome on a wider spatial scale is needed for a better understanding of the terroir of each coffee planting region. Therefore, our aim was to evaluate the microbial communities harbored in soils and fruits of these Coffea species in four Brazilian floristic domains (Amazon, Atlantic Forest Caatinga, and Cerrado). One hundred and eight samples (90 of soil and 90 of fruits) were used in the extraction and sequencing of the fungal and bacterial DNA. We detected more than 1000 and 500 bacterial and fungal genera, respectively. Some soil microbial taxa were more closely related to one coffee species than the other species. Bacillus bataviensis tends to occur more in arid soils from the Caatinga, while the fungus Saitozyma sp. was more related to soils cultivated with C. arabica. Thus, the species and the planting region (floristic domain) of coffee affect the microbial composition associated with this crop. This study is the first to report microbial communities associated with coffee produced in four floristic domains that include sites in eight Brazilian states. Data generated by DNA sequencing provides new insights into microbial roles and their potential for the developing more sustainable coffee management, such as the production of biofertilizers and starter culture for fermentation of coffee cherries.

Coffea arabica and Coffea canephora are the main species of coffee cultivated in Brazil and worldwide.C. canephora is cultivated at altitudes ranging from 50 to 550 m above see level (asl) and is responsible for about 25% of the total national production of coffee.C. arabica represents about 75% of the total production and is grown at higher altitudes (600-1200 m asl) 1 .
Many factors contribute to the success of coffee production.These include pre-and post-harvest factors.Among the pre-harvest factors, the microbiota associated with fruits and soils are key drivers to achieve highquality production.In soils, for example, arbuscular mycorrhizal fungi (AMF) and nitrogen-fixing bacteria can improve the nutrient uptake by plants by delivering reduced nitrogen and solubilizing phosphate, otherwise unavailable to plants 2 .Appropriate crop management can improve the effect of these beneficial microorganisms on plant growth and allow coffee producers to minimize the use of chemical fertilizers and pesticides, which has been an increasing demand of the market and has environmental impacts 3 .
The strategy of revealing the microorganisms associated with soil and fruits of coffee species is a crucial step to allow the prospecting of beneficial microorganisms that can be used as biofertilizers in soils and starter cultures for improving coffee beverage quality 4,5 .The coffee cultivars are more adapted to some regions than others and

Microbial diversity
The bacterial communities inhabiting fruits and soils of coffee plants differ between the host species (C.arabica vs. C. canephora, Fig. 3A).This difference is greater between the species than among the floristic domains (see F-statistics in Fig. 3B).By the PERMANOVA p-values, the fungal communities in coffee fruits were about two times (17.832/6.752= 2.64) higher than the bacterial communities.Nonetheless, in soils the divergence was more than four times (17.8329/6.752= 4.41) greater for the fungal community.These results show that the rhizospheric mycobiome is very different between the coffee species (Fig. 3, Fig. S1).
Besides the difference between the microbiomes of the two coffee species (Fig. 3B), the bacterial communities from the Atlantic forest, i.e.Araponga (site AW) and São Paulo (site AV), had a distinct community composition (Fig. 4A).In addition, the fungal communities associated with C. arabica of the Caatinga were distinct from those of other locations (Fig. 4B).This result may be due to terroir of the Caatinga floristic domains, which had low annual precipitation, high temperatures throughout the year, and shallow and stony soil.Regardless of the alpha metrics between species, the richness, evenness, and diversity of soil bacteria were higher than the fungal measurements, while in fruits the bacterial richness was lower than that of fungi (Fig. 5A).This same pattern was also observed across the floristic domains (Fig. 5B).However, it is worth noting that most of the sequencing effort in fruits was lost by the amplification of sequences from mitochondria and chloroplasts, therefore the observations of the bacteria in fruits should be done with caution.We did not find significant differences in the alpha diversity metrics among species.As expected, the highest values of diversity were observed in the coffee plantation under organic management (property AW, Fig. 6) and the lowest values of bacterial diversity were found in plantations where extractivism without management is performed (property AI).We also found that the fungal diversity in soils and fruits were inversely correlated (r pearson = − 0.37; p-value = 0.045).
Some specific taxa have shown a distinct relative abundance related to the floristic domain or to the coffee species.For instance, the genus Saitozyma displayed higher relative abundances in soils of C. arabica (mean relative abundance = 4.13% ± 3.56) than in soils of C. canephora (0.17% ± 0.23) (Fig. S2).Furthermore, Bacillus bataviensis had the highest relative abundance in the soils of the Caatinga floristic domain (Properties AI, AK, and AM; Fig. S3).The most abundant nitrogen-fixing bacterial group found in the soils of C. arabica was the Bradyrhizobium genus (Proteobacteria Phyla, Fig. S3) while in C. canephora, Sphingomonas was the main genus found.As shown before (Fig. 3A), fungal composition differed greatly between the coffee species.The main fungi found in both species could not be identified to deeper levels of classification (i.e.species, genus, and families).
We detected 356 Amplicon Sequence Variants (ASVs) from the phylum Glomeromycota that are Arbuscular Mycorhiza Fungi (AMFs) (Fig. S4).These were clustered into 191 operational taxonomic units (OTUs).Most OTUs could not be identified to the genus or species level, with most of them being identified only to the order or family level.Furthermore, only ten OTUs of AMFs were shared between both Coffea species, and the remaining were associated with only one coffee species.Some of these OTUS were observed exclusively in locations with water shortages.

Discussion
This study is the first to report microbial communities associated with Brazilian coffee produced in four floristic domains (Amazon, Atlantic Forest, Caatinga, and Cerrado) and eight states (PR, SP, MG, ES, BA, CE, PE, and RO).The amount of data generated by DNA sequencing was enough to assess the microbial diversity in fruits and soils of coffee plants (Supplementary Table 1), while the large amount of data provides new insights into microbial roles and their potential for the development of more sustainable coffee management.For instance, The distinct microbial community associated with each coffee species and floristic domain observed in this study (Fig. 3) may be due to the rhizosphere effect of the host.However, we cannot consistently assert that the differences found in both communities were exclusively due to the Coffea species because this factor is mixed up with the environmental conditions under which each species grows.For instance, the cultivation altitudes of the two coffee species are different.C. arabica is cultivated at high altitudes, while C. canephora plantations are usually located at low altitudes.The samples of this study were obtained at altitudes below 553 m for C. canephora and above 763 m for C. arabica.It is interesting to note that the microbial communities associated with the rhizosphere of C. canephora at the highest elevation (553 m) were more similar to the microbial communities associated with the rhizosphere of C. arabica at the lowest elevation (763 m).Furthermore, these two crops are located close to each other (approximately 18 km) and other studies have shown that altitude is one of the main factors driving the microbial composition of coffee soils 6,11 .Moreover, other factors like the management system 12 , the phenological stages of coffee 12,13 and the chemical composition of soil can also modulate the edaphic microbial community 6 .
The high relative abundances of Bacillus bataviensis in the soils from the Caatinga (Properties AI, AK, and AM) may be due to the characteristics of adaptations and survival of this microorganism in soils with low water availability.The increased relative abundance of this genus was related to increased plant resilience during drought stress [14][15][16] .The Caatinga region has low pluviometric indices and the presence of soil microorganisms that improve plant resilience may be one of the keys that enables the growth of coffee plants under these harsh conditions.Furthermore, the relatively high abundance of Saitozyma in soils of C. arabica may be related to the content of organic matter.This fungus has the potential to degrade plant-derived lignocellulosic compounds in the soil and carbohydrates with five carbon atoms 17 .According to Aliyu et al. 17 , in the Saitozyma sp.growth on D-xylose there is an overrepresentation of genes for the D-xylulose reductase/L-iditol 2-dehydrogenase.The degradation of xylose produces xylitol, which is used as raw material in the sweetener production with a low glycemic index.Thus, the Saitozyma sp. has potential to be used as starter culture in coffee fermentation to increase the sensory perception of sweetness in the coffee beverage.
One of the most abundant bacterial ASVs able to fix nitrogen observed in both Coffea species was Bradyrhizobium sp. (Fig. S3).This result corroborates recent studies of the microbiome associated with the soils of coffee plants 18,19 .Furthermore, other genera, for example Sphigomonas and Sphingobium (Fig. S3) related to nitrogen fixation are present in soils of C. arabica 20 .However, there are still no commercial inoculants of nitrogen-fixing bacteria for coffee planting, which shows the potential for bioprospecting of the native coffee microbiota.
A high number of ASVs (191 OTUs at 97% threshold) were identified as belonging to the Glomeromycota (Fig. S4).However, it is important to note that the relative abundance of these fungi was below one percent in samples.Although ITS1F/ITS4 are often a suitable pair of primers to estimate the overall fungal diversity of samples, some authors argue that this primer set used in DNA amplification has a great impact on the diversity of AMF detected by the Next Generation Sequencing 21,22 .The AMFs have an important role in plant growth and yield because they can improve soil fertility and water and nutrient uptake by plants 2 , which is very important in coffee production under conditions of water stress like those observed in Caatinga soil.Furthermore, the presence of these fungi has been shown to benefit from agroecological management 12 which is mostly due to the reduction in the amount of the chemical fertilizers used.
The area with organic management (AW) had the highest fungal diversity (Fig. 6) and the most distinct bacterial beta diversity (Fig. 4) among the 30 plantations evaluated in this study.Variations in microbial diversity were greater for the fungal community than for other microbial communities under different types of management 12,23 .The use of conventional agrochemical management may reduce microbial diversity due to the toxic effects of these products 24 .However, the lowest bacterial diversity was observed in coffee planted within forests under extractivism without management (site AI, Fig. 6).The lack of management in extractivism may compromise the condition of the soil, leading to negative impacts on bacteria.Therefore, crop management has a high impact on soil microbial diversity.
Because of the distinct fungal communities harbored by fruits of each coffee species (Fig. S3), we investigate if these species could harbor yeasts with potential for use as starter culture in the fermentation of coffee fruits.Coffee fermentation has been used to improve the chemical and sensory quality of C. arabica and C. canephora fruits 4,25,26 .In this technique, a starter culture (e.g.Saccharomyces cerevisiae) is added into the fermentation tank containing fruits or beans of coffee 4,25 .Although most of ASV yeasts have not been identified to the species level, some of them are found exclusively in one of the two coffee species or in only one of the floristic domains (Fig. S5).This result highlights the importance of testing wild yeasts of coffee cherries in fermentation as these microorganisms may be more adapted to the natural condition of the coffee-growing region and provide better Figure 6.Alpha-metrics of bacterial (left plot) and fungal (right plot) communities harbored in fruits and soils of coffee plants from two species: Coffea arabica (green) and Coffea canephora (red).The letters above each boxplot were calculated using the Kruskal-Wallis' test.Areas with the same letter do not have statistical differences.
flavors to the beverage.The use of wild yeasts in fermentation of fruits other than coffee have shown potential for producing beverages with more desirable sensory attributes than those of the commercial strains 26 .
This broad survey performed across the Brazilian coffee producing regions showed that the genus Coffea harbors a high diversity of microbial communities associated with fruits and soil.There is a difference between the microbial communities associated with C. arabica and C. canephora in each floristic domain.Furthermore, within each species there is also variation among the microbial communities in the planting region.Many fungi and bacteria detected that have not been cultivated yet show potential for application in pre-and post-harvesting of coffee production (Figs.S4 and S5).Nitrogen-fixing bacteria and AMFs may be used in pre-harvesting to increase plant growth and fruit production, while the yeasts and bacteria had potential to be applied in coffee fermentation.Therefore, this opens up an opportunity to better explore the microbial potential for the development of a sustainable coffee production chain.

Study area
A total of 180 samples (90 from soil and 90 from fruits) were collected from 30 plantations (seven of C. canephora and 23 of C. arabica; Table 1) of eight Brazilian states located in four floristic domains (Amazon, Atlantic Forest, Cerrado, and Caatinga; Fig. 1).The sampling process was performed as described by Veloso et al., 2020.

DNA extraction and PCR
Aliquots of 250 mg of each sample were added to NucleoSpin ® Bead Tubes Type A (containing ceramic beads) with 700 μL of SL1 buffer and 150 μL of Enhancer SX provided by the kit Nucleospin Soil ® (Machanarey-Nagel) 27 .
Cell lysis was carried out in a Precellys 24 High-Powered Bead Mill Homogenizer (Bertin technologies) for 50 s at 4000 rpm.DNA extraction was performed according to the manufacturer's protocol.The quality of DNA extraction was evaluated by electrophoresis in 0.8% agarose gel stained with ethidium bromide under UV light.The region V3-V4 of 16S rDNA gene was amplified with the primers 341F (5'-CCT ACG GGA GGC AGCAG-3') and 806R (5'-GGA CTA CNVGGG TWT CTAAT-3') 28 .The Internal Transcribed Spacer 1 (ITS1) fungi were amplified with the primers ITS1F (5'-CTT GGT CAT TTA GAG GAA GTAA-3') and ITS2 (5'-GCT GCG TTC TTC ATC GAT GC-3') 29 .The PCR reactions were performed with Phusion ® High-Fidelity PCR Master Mix (New England Biolabs) and specific barcodes were used for each sample.The PCRs were evaluated in a 2% agarose gel using SYBR green.Samples with the main bright strip between 400 and 450 bp were used for downstream experiments.The PCR were mixed in equally dense ratios and purified with Qiagen Gel Extraction Kit (Qiagen, Germany).
The sequencing libraries were prepared using NEBNext ® Ultra™ DNA Library Prep Kit for Illumina according to the manufacturer's recommendations.The library quality was evaluated on a Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system.Finally, the Illumina NovaSeq 6000 platform was used to sequence the libraries to produce 250 bp paired-end reads.

Bioinformatic analyses
The raw reads were demultiplexed and trimmed to remove primers and adapters.All those with a maximum number of expected error (maxee) equal or greater than one and the chimeras or singletons were removed.

Figure 2 .
Figure 2. Overview of the sequencing number obtained by sequencing of the bacterial and fungal communities from soil and fruits of C. arabica and C. canephora.

Figure 3 .
Figure 3. Beta-diversity of the whole community (bacteria + fungi; left plot), bacterial (middle plot) and fungal (right plot) communities harbored in fruits and soils of coffee plants from two species: Coffea arabica and Coffea canephora.The upper panel (A) shows the difference between microbial beta-diversities associated with the soil of each coffee species (C.arabica or C. canephora), while the lower panel (B) represents the differences among floristic domains.The PERMANOVA F-statistics were calculated based on 999 permutations.The higher the value of F-statistics, the greater the difference of microbial communities between (A) coffee species or (B) floristic domains.

Figure 4 .
Figure 4. Beta-diversity of (A) fungi and (B) bacteria found in soil of C. arabica and C. canephora plantations.Each plantation is represented by three points connected by dashed lines.Each point represents the positions in the two first coordinates of the Principal Coordinate Analysis (PCoA) built from the Bray-Curtis distance matrix.

Figure 5 .
Figure 5. Alpha-metrics of whole community (bacteria + fungi; left plot), bacterial (middle plot) and fungal (right plot) communities harbored in fruits and soils of coffee plants from two species: Coffea arabica and Coffea canephora.The upper panel (A) shows the difference in microbial alpha diversity metrics between coffee species (C.arabica or C. canephora), while the lower panel (B) represents the differences among floristic domains.The PERMANOVA F-statistics were calculated based on 999 permutations.The higher the value of F statistics, the greater the effect of the species or floristic domains on the community.

Figure 7 .
Figure 7. Taxonomic composition at the phylum level in soils of each coffee plantation.Values within bars represent the relative abundance of each phylum based on the 16S and ITS1 sequencing.

Table 1 .
Coffee farms in Brazil (30 farms) used in this study to evaluate the coffee microbiome.A total of three samples per farm was used for all the analyses.